Heterologous production of 10-methylstearic acid

Abstract

Nucleic acids and cells comprising a methyltransferase gene and/or a reductase gene are disclosed. These nucleic acids and cells may be used to produce branched (methyl)lipids, such as 10-methylstearate.

1 . A yeast cell comprising a methyltransferase protein encoded by a methyltransferase gene and either a branched (methyl)lipid or an exomethylene-substituted lipid, wherein:
 the methyltransferase protein comprises an amino acid sequence with at least 90% sequence identity with the amino acid sequence of SEQ ID NO:76;   the branched (methyl)lipid or exomethylene-substituted lipid is a carboxylic acid, carboxylate, ester, thioester, or amide; and   the branched (methyl)lipid comprises a saturated or unsaturated branched aliphatic chain comprising a branching methyl group or the exomethylene-substituted lipid comprises a branched aliphatic chain that is branched because the aliphatic chain is substituted with an exomethylene group.   
     
     
         2 . The yeast cell of  claim 1 , wherein the branched (methyl)lipid or the exomethylene-substituted lipid comprises a linear lipid with a chain length of 14-20 carbons and a methyl branch at the Δ9, Δ10, or Δ11 position. 
     
     
         3 . The yeast cell of  claim 2 , wherein the branched (methyl)lipid or the exomethylene-substituted lipid is a diacylglycerol, triacylglycerol, or phospholipid, and the diacylglycerol, triacylglycerol, or phospholipid comprises an ester of 10-methylstearate or an ester of 10-methylenestearate. 
     
     
         4 - 6 . (canceled) 
     
     
         7 . The yeast cell of  claim 1 , wherein at least 1% by weight of the fatty acids of the yeast cell are one or more linear fatty acids with a chain length of 14-20 carbons and a methyl branch at the Δ9, Δ10, or Δ11 position. 
     
     
         8 . The yeast cell of  claim 1 , wherein the yeast cell comprises at least 1% lipid as measured by % dry cell weight. 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The yeast cell of  claim 1 , further comprising a recombinant reductase gene. 
     
     
         13 . The yeast cell of  claim 12 , wherein the recombinant reductase gene encodes tmsA from  Thermomonospora curvata.    
     
     
         14 . The yeast cell of  claim 1 , wherein:
 the methyltransferase gene is codon-optimized for the yeast cell; or   the yeast cell further comprises a reductase gene and the reductase gene is codon-optimized for the yeast cell.   
     
     
         15 - 19 . (canceled) 
     
     
         20 . The yeast cell of  claim 1 , wherein the yeast cell is selected from the group consisting of  Arxula, Saccharomyces , and  Yarrowia.    
     
     
         21 . The yeast cell of  claim 20 , wherein the yeast cell is selected from the group consisting of  Arxula adeninivorans, Saccharomyces cerevisiae,  and  Yarrowia lipolytica.    
     
     
         22 - 23 . (canceled) 
     
     
         24 . The yeast cell of  claim 1 , wherein the methyltransferase gene comprises either:
 a nucleotide sequence with at least about 95% sequence identity with the nucleotide sequence of SEQ ID NO:75; or   the nucleotide sequence of SEQ ID NO:75.   
     
     
         25 - 28 . (canceled) 
     
     
         29 . A method of producing a branched (methyl)lipid or exomethylene-substituted lipid, comprising contacting the yeast cell of  claim 1  with oleic acid, methionine, or both oleic acid and methionine. 
     
     
         30 - 49 . (canceled) 
     
     
         50 . The yeast cell of  claim 12 , wherein the reductase protein is in a fusion protein with the tmsB enzyme.